The effect of the pH level of the substrate on the rate of cellular

respiration in Yeast (Saccharomyces cerevisiae)

BIO 11.1 22L

November 27, 2018

______________
A scientific paper submitted in partial fulfillment of the requirements in BIO 11.1,
1st Semester, 2018-2019 under Prof.
 Abstract
The study was conducted in order to find out the effect of the pH
level of the substrate on the rate of the cellular respiration of yeast. This
was done by preparing four set-ups with three replicates. Yeast suspension
and glucose solutions with different pH levels of equal amount were
added into each tube. After the tubes were sealed, the height of carbon
dioxide formation in each tube was recorded. The data recorded were
used to compute for the volume of CO₂ production and the rate of
cellular respiration. The results showed that the set-up 1 with the acidic
solution exhibited the highest volume and fastest rate of carbon dioxide
formation followed by set-up 3, set-up 2, and set-up 4 respectively. This is
due to the higher concentration of H⁺ ions in the solution which bonded
with the anionic regions of the enzymes in the yeast that resulted to a
faster cellular respiration rate. Thus, it can be concluded that yeast exhibit
optimal cellular activities in an acidic environment than in neutral and
basic ones.

Introduction
Cellular respiration is the catabolic breakdown of organic compounds

with the help of enzymes in order to release the stored energy in the form of

Adenosine triphosphate (ATP) and use it to fuel an organism (Urry et al. 2017).

Cellular respiration can either be aerobic or anaerobic, one process requiring

oxygen and the other not respectively. During fermentation, the products of

photosynthesis are converted to carbon dioxide and energy without requiring

oxygen (Burgess & Pletschke n.d.). Fermentation has two types: the lactic acid

fermentation which occurs on animals and produces lactic acid and alcoholic

fermentation which occurs on plants and microorganisms and produces ethanol

(Campbell et al. 2006).

Saccharomyces cerivisiae is specie of yeast capable of both aerobic and

anaerobic respiration that usually uses glucose for its substrate (Reece et al.

2011). It undergoes glycolysis which is a breakdown of one molecule of glucose

into two molecules of pyruvate, an organic compound with a backbone of the

carbon atoms.

Glucose is the most essential simple sugar in the metabolism of a lot of

organisms. It is called a monosaccharide which is the building block of all

carbohydrates (McMurry 2012). It serves as fuel in generating energy that the

body cells utilize in order to carry out cellular activities like cellular respiration. The

production of energy under alcoholic respiration of glucose in yeast is presented

in Figure 1.1:

Figure 1.1. The process of Alcoholic Fermentation of glucose in yeast

Different factors can affect anaerobic respiration in yeast. These different

factors are the temperature, the nutrient availability, the substrate used, and the

pH level of the yeast’s environment. According to Ai et al. (2015), yeasts have a

high acidity tolerance that enables them to carry out optimal biological

processes at a low pH level ranging from pH 4 to pH 4.5.

 In order to further understand the effect of the pH condition of the

substrate to the rate of cellular respiration in yeast, a study was conducted. If the

optimum pH level for yeast cellular respiration is around 4.5 then the yeast in the

acidic solution will exhibit a faster rate of carbon dioxide production than the

yeast in the neutral and basic solutions. Specifically, the experiment aims to

pinpoint the optimal pH condition in which the yeast will exhibit a faster

respiration rate and determining if there will be a significant change on the

medium’s pH level after the yeast utilized the glucose present in the solution for

respiration.

Determining the effect of the pH level on the rate of cellular respiration in

yeast can offer additional understanding of the concept of cellular respiration. It

may also serve as a reference for further studies on the subject and for

experiment modification to future researchers.

The study was conducted on the 8th of November, 2018 at Wing C Room

117 of the Institute of Biological Sciences in the University of the Philippines, Los

Baños.
 Materials and Methods

To determine the effects of the pH level on the cellular respiration rate of

yeast, four set-ups with three replicates in Smith fermentation tubes were

created. Water with the pH level of 9 and 7 were bought from the convenience

store while the water with a pH level of 4 was prepared through the titration of

one drop of 1 N HCl onto water with a neutral pH level. Then, the 10% glucose

solution was prepared by combining 4.5 grams of glucose powder and 45 mL of

water with different pH levels in three separate beakers. Ten milliliters of the

glucose solution was poured into the tubes; pH 4 – set-up 1, pH 7 – set-up 2, pH 9

– set-up 3. Five milliliters of the yeast suspension, prepared by adding six grams of

yeast into 60 mL of distilled H₂O, was also added into each tube. The tubes were

sealed with cotton plugs after making sure that there were no trapped air

bubbles inside of the tubes’ vertical arms.

The fourth set-up served as the negative control in which only five milliliters
of yeast suspension and ten milliliters of distilled water were added into each
tube.

Figure 2. The set-up for measuring the CO₂ production of yeast

 Then, the height of the carbon dioxide produced in each tube was

measured. This was done for 45 minutes at 15-minute intervals.

The volume of the production of carbon dioxide was computed with the

formula:

𝑉𝑜𝑙𝑢𝑚𝑒 (𝑐𝑚3 ) = 𝜋𝑟 2 ℎ

Where: r = radius of the vertical arm

h = height of the space occupied by the carbon dioxide

The average volume of the carbon dioxide production from the four set-

ups were computed and recorded in Table 1.1.

To determine the partial rate of CO2 production with respect to given

interval, the formula shown below is used:

𝑉𝑖 − 𝑉𝑖−1
𝑟𝑝 =
𝑡𝑖 − 𝑡𝑖−1

Where: Vᵢ = volume of the CO₂ at a given time

Vᵢˍ₁ = volume of CO₂ immediately before Vᵢ

t ᵢ = time when Vᵢ was measured

tᵢˍ₁ = time immediately before tᵢ

The computed values were illustrated in a line graph in Figure 3.1.

The average rates of carbon dioxide production for each substrate were

computed through the formula:

𝑐𝑚3 𝑓𝑖𝑛𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒

𝑟𝑎 ( )=
𝑚𝑖𝑛 𝑇𝑜𝑡𝑎𝑙 𝑡𝑖𝑚𝑒

The data from this computation were illustrated in Figure 3.2.

 To find out if there will be a significant change in the pH level of the set-

ups after 45 minutes, the initial and final pH values were recorded in Table 1.3.
 Results and Discussion

Table 1.1. Average Volume (cm³) of CO₂ Production in 4 set-ups recorded within 45
minutes at five-minute intervals

Time (mins) Volume of CO₂ Produced (cm)

Set-up 1 Set-up 2 Set-up 3 Set-up 4

0 0.00 0.00 0.00 0.00
15 3.31 2.96 3.52 0.00
30 8.92 7.21 5.26 0.10
45 12.17 10.75 11.81 0.61

The highest amount of carbon dioxide production was constantly

exhibited by set-up 1 with the acidic solution, followed by set-up 3 with the basic

solution, then set-up 2 with the neutral solution. This means that the yeast in set-

up 1 was able to utilize the glucose in the solution more than the yeast in the

neutral and basic solutions. As said by Ai et al. (2015), yeast was able to carry out

optimal cellular activities at the pH of 4 to the pH of 4.5.

The negative control, set-up 4, also exhibited carbon dioxide production

albeit at significantly lower amount. This is due to the ability of the yeast to utilize

their glycogen reserves during a long-term nutrient deprivation (Wilson et al.

2010).
 0.4

Partial Rate CO₂ Production

0.35
0.3

(cm³/min)
0.25
pH 4
0.2 pH 7
0.15 pH 9
NC
0.1
0.05
0
0 15 30 45
Time (min/s)
Figure 3.1. Partial Rate (cm³/min) of CO₂ Production in 4 set-ups recorded within 45
minutes at five-minute intervals
0.28
Average Rate of CO₂ Production (cm³/min)

0.26
0.24
0.22
0.2
0.18
0.16 Acidic
0.14
Neutral
0.12
0.1 Basic
0.08
0.06
0.04
0.02
0
Set-ups

Figure 3.2. Average rate (cm³/min) of CO₂ production in 4 set-ups recorded in 45 minutes.

Figure 3.1 and 3.2 shows that during the period of 45 minutes, set-up 1 with

the acidic condition has the fastest rate of 2.70 x 10-1 cm³/min among the four

set-ups. Set-up 1 is followed by set-up 3 – basic solution and set-up 2 – neutral

solution with 2.62 x 10-1 cm³/min and 2.39 x 10-1 cm³/min respectively. In a study

done by Batstone et. al (2016), a lower pH value also means that the solution has

a higher concentration H⁺ ions. These ions form covalent bonds with the freely

exposed anionic regions on the surface of the enzymes which speeds up the

process of cellular respiration (Batstone et al. 2016).

 Also, there is noticeable decline on the rate of cellular respiration in the

first three set-ups after the 30-minute mark. According to a study Stanley et al.

(2009), ethanol accumulation during yeast respiration reduces cell vitality and

increase cell death. A high concentration of ethanol also decrease the water

availability in the yeast cells, this causes the inhibition of key glycolytic enzymes

thus slowing down the cellular respiration in yeast. (Stanley et al. 2009).

Table 1.2. Change in pH level of the solutions in the four set-ups after 45 minutes of yeast
respiration

Set-up Initial pH Final pH

Acidic 4.3 4.4

Neutral 7.3 4.4

Basic 9.4 4.5

Negative Control 6.9 5.5

From the initial pH levels, there is a significant change on the final pH level

of the solutions except for the acidic set-up. As the yeast consumed the glucose,

ethanol and organic acids are produced. Also, the addition of carbon dioxide

contributed on the acidity of the solutions after the alcoholic fermentation.

The solutions also seem to reach only up to the optimal pH level of

approximately 4.5. According to Barrio et al. (2009), if the pH of the solution

further decreased lower than the optimal condition, then the denaturation of

the yeast will instead take place and their metabolism will slow down.
Figure 3.3. Smith fermentation set-up with undergoing carbon dioxide production
 Summary and Conclusion

The study aims to assess how the pH level of the medium affects the rate

of cellular respiration in Saccharomyces cerevisiae. Its goal is to find out if having

a lower pH level of around 4.5 will result to faster rate of cellular respiration.

Specifically, the study will focus on pinpointing the optimal pH condition in

which the yeast exhibit a higher respiration rate; and determining if there is a

significant change on the medium’s pH level after the yeast utilized the glucose

present in the solution for anaerobic respiration.

The study was done by preparing four set-ups, each with three replicates.

Each tube from the first three set-ups was filled with five milliliters of yeast

suspension and ten milliliters of 10% glucose solution. The pH level of the water

used was different for every set-up; pH 4 for set-up 1, pH 7 for set-up 2, and pH 9

for set-up 3. Set-up 4 served as the negative control, having only the yeast

suspension and water. Before the period of 45 minutes, the pH levels of the

solutions were recorded. Then, the height of CO₂ produced was measured at 15

minute intervals for 45 minutes and was used to compute the rate of cellular

respiration for each set-up. After the yeast respiration, the final pH levels of the

solutions were then recorded.

Based on the recorded data, Set-up 1 with pH 4 medium exhibited the

fastest rate of cellular respiration, followed by set-up 3 with pH 9 medium then

set-up 2 with pH 7. Set-up 4 also exhibited cellular respiration but on a

significantly slower rate than the first three set-ups due to the lack of substrate.

After 45 minutes, all of the set-ups exhibited change in their pH levels.

The pH level of the solutions in the set-ups exhibited a close yet observable

effect on the cellular respiration of the yeast in the glucose solutions. Hence, the

effect of the pH level of the solution to the fermentation process of the yeast was

positively confirmed in the experiment. The acidic set-up produced the highest

amount of carbon dioxide and exhibited the fastest rate of cellular respiration

among the four set-ups.

Therefore, the yeast exhibit optimal utilization of carbon sources in a more

acidic environment over a basic and neutral environment.

To address the results obtained from set-up 2 and 3, reevaluation of the

methodology must be done in order to attain a more reliable pattern of the

yeast’s pH preference. It is recommended that future researchers prepare

multiple set-ups with a wider range of pH values. Also, the usage of larger Smith

fermentation tubes is recommended. To keep the concentration of the yeast

suspension constant for each set-up, uniform stirring is recommended before the

inoculation of the yeast into the glucose solutions.

On the other hand, for further study and understanding of the change in

the pH level of the solutions after the yeast cellular respiration, recording data at

shorter time intervals is recommend in order to obtain substantial amount of data

for the research.

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